人组织激肽释放酶成熟蛋白在大肠杆菌中的高效表达

将编码人组织激肽释放酶成熟蛋白的基因片段扩增并分别克隆到原核表达载体pET2 8(b)及分泌型表达载体pET2 0 (b)中 ,使其C端融合 6×HisTag序列 .转化不同受体菌 ,IPTG诱导表达后利用SDS PAGE、免疫印记等方法对重组蛋白进行分析 .在 6株基因工程菌株中 ,均表达出分子量约30kD的激肽释放酶融合蛋白 ,其中激肽释放酶在pET2 8载体中的表达水平高于pET2 0载体 .pET2 8和pET2 0载体表达的重组激肽释放酶蛋白分别占菌体总蛋白约 2 6 %和 10 % .Western印迹分析表明 ,目的蛋白可与抗人血清KK单克隆抗体发生特异性反应 .未经纯化的激肽释放酶融合蛋白具有一定的水解苯甲酰精胺酸乙酯 (BAEE)的能力 .在大肠杆菌中获得了人组织激肽释放酶的高效表达 ,表达产物具有免疫原性和生物活力 ,这为研究其生物功能和开发基因工程药物奠定基础     

Preserved functional specialization for spatial processing in the middle occipital gyrus of the early blind

The occipital cortex (OC) of early-blind humans is activated during various nonvisual perceptual and cognitive tasks, but little is known about its modular organization. Using functional MRI we tested whether processing of auditory versus tactile and spatial versus nonspatial information was dissociated in the OC of the early blind. No modality-specific OC activation was observed. However, the right middle occipital gyrus (MOG) showed a preference for spatial over nonspatial processing of both auditory and tactile stimuli. Furthermore, MOG activity was correlated with accuracy of individual sound localization performance. In sighted controls, most of extrastriate OC, including the MOG, was deactivated during auditory and tactile conditions, but the right MOG was more activated during spatial than nonspatial visual tasks. Thus, although the sensory modalities driving the neurons in the reorganized OC of blind individuals are altered, the functional specialization of extrastriate cortex is retained regardless of visual experience.     

不同浓度碘对大鼠仔鼠垂体促甲状腺激素细胞的影响

目的探讨不同浓度碘对60日龄仔鼠垂体TSH细胞的影响。方法将断乳1个月的Wistar大鼠仔鼠,雌雄各半随机分为5组:NI组、10HI组、50HI组、100HI及LI组。饲养3个月的雌雄大鼠1∶1合笼交配产生仔鼠,断乳后的仔鼠喂养同上述大鼠。测定60日龄仔鼠垂体TSH细胞的体密度、强阳性细胞百分数及甲状腺的相对重量、血清甲状腺激素水平。结果50HI、100HI组仔鼠垂体TSH细胞的体密度、强阳性细胞百分数及血清T4明显高于NI组,LI组垂体TSH细胞的体密度明显高于NI组、血清T4明显低于NI组,各碘过量组甲状腺的相对重量与NI组相比没有差别,LI组仔鼠甲状腺的相对重量明显高于NI组。结论碘缺乏对甲状腺、垂体TSH细胞的影响要强于碘过量对甲状腺、垂体TSH细胞所造成的影响。     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.     

雌激素Beta受体在大鼠脑内表达的免疫组化定位研究

为了探讨雌激素作用于神经系统的机理,采用硫酸镍铵增强显色的免疫组化SP法研究了新的雌激素受体(ER-β)在成年雌雄大鼠脑内的分布。研究证实ER-β免疫阳性物质主要位于神经元的细胞核内,但在个别脑区也可在胞浆甚至突起内检测到。最强的ER-β免疫阳性信号见于前嗅核、大脑皮质、小脑浦肯野细胞、斜角带垂直部、蓝斑和三叉神经运动核等部位;中等强度的染色见于隔内侧核、杏仁外侧核、黑质、中央灰质等部位;较弱的阳性反应见于下丘脑与杏仁复合体的部分核团。在一些部位还存在表达水平甚至细胞内定位模式的性别差异,如前庭上核内的表达只见于雌性;雄性大鼠三叉神经运动核内ER-β蛋白主要表达于胞浆内,细胞核为阴性;而在雌性大鼠该部位ER-β蛋白主要位于细胞核等。以上结果表明ER-β蛋白在大鼠脑内分布广泛并具有一定的性别差异,在与学习记忆有关的脑区如大脑皮质和基底前脑内有很高的表达,提示在脑组织内雌激素可能通过ER-β这一新的信号途径发挥多种重要的调控作用,如学习记忆等。     

用超声法从花锚中提取(口山)酮类化合物

用超声法从高原植物椭圆叶花锚的全草中,提取并分离出2种针状结晶化合物;采用元素分析(EA)、核磁共振波谱(NMR)、质谱(MS)、红外光谱(IR)、紫外光谱(UV)、熔点测定等分析方法,对其化学结构进行表征;产物分别为1,3-二羟基-4,5,8-三甲氧基酮和1-羟基-2,3,4,8-四甲氧基酮。     

玉米赤霉烯酮在冬小麦短日春化中的作用

应用酶联免疫技术,研究了冬小麦品种燕大１８１７ 在不同光周期条件下,体内内源玉米赤霉烯酮（ＺＥＮ） 和ＡＢＡ 含量的变化。结果表明,冬小麦品种燕大１８１７ 具有短日春化性,苗期经４ 周ＳＤ 处理后转到ＬＤ 下能正常抽穗。ＳＤ 诱导春化的幼苗茎尖和叶片中ＺＥＮ 含量高于未经诱导的ＬＤ 幼苗,而在两种光周期条件下生长的幼苗中ＡＢＡ 含量变化趋势并无明显差异。马拉硫磷（ ＭＡＬ） 抑制ＳＤ 幼苗体内ＺＥＮ 含量高峰的出现,也部分抑制抽穗;外源ＺＥＮ 可部分抵消ＭＡＬ对植株的影响,降低茎尖内源ＡＢＡ 含量,并有促进小麦拔节的作用。说明在冬小麦短日春化过程中,ＺＥＮ 具有促进作用,而ＡＢＡ 没有直接影响。     

Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet (“Western blot”). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.